blood gases, and extracellular/intracellular acid … · acid-base status and temperature in...

J. exp. Biol. 130, 13-25 (1987) 13Printed in Great Britain © The Company of Biologists Limited 1987

BLOOD GASES, AND EXTRACELLULAR/INTRACELLULARACID-BASE STATUS AS A FUNCTION OF

TEMPERATURE IN THE ANURAN AMPHIBIANSXENOPUS LAEVIS AND BUFO MARINUS

BY R. G. BOUTILIER*, M. L. GLASSf AND N. HEISLER

Abteilung Physiologie, Max-Planck-Institut fur experimentelle Medizin,Gottingen, FRG

Accepted 4 March 1987

SUMMARY

Blood gases, and parameters of the extracellular and intracellular acid-base status,were measured in the anuran amphibians Bufo marinus and Xenopus laevisacclimated to temperatures of 10, 20 and 30°C for 12 days. Arterial POz rose withtemperature so that approximately constant oxygen saturation of the blood wasmaintained, a phenomenon explained on the basis of models for O2 transport inanimals with central vascular shunts and temperature-dependent shifts in O2

equilibrium characteristics. Arterial plasma pH of both species varied inversely withtemperature, the pH/temperature coefficient being not significantly different fromthat required for constant relative alkalinity or dissociation of imidazole. The changein plasma pH was brought about mainly by changes in Pco2> although plasmabicarbonate concentration also changed significantly. Intracellular pH/temperaturerelationships were found to be non-linear in most of the tissues. There wasconsiderable variability among body tissue compartments and between the twospecies. These data confirm that the various tissue compartments in ectothermsmaintain unique ApH/At relationships, and indicate that measurement of extra-cellular pH as a function of temperature is not a good indicator for alphastat-type,temperature-dependent, acid—base regulation.

INTRODUCTION

The early studies on the inverse relationship between extracellular pH and bodytemperature in ectothermic vertebrates (e.g. Robin, 1962; Rahn, 1967; Howell,Baumgardner, Bondi & Rahn, 1970; for further references see Heisler, 19866) led tothe formulation of two models: that of a constant relative alkalinity (Rahn, 1967) andthe alphastat hypothesis (Reeves, 1972). These models were based on experimental

•Present address: Department of Biology, Dalhousie University, Halifax, Nova Scotia,B3H4J1, Canada.

f Present address: Department of Zoophysiology, University of Aarhus, DK-8000 Aarhus,Denmark.

Key words: acid-base regulation, Amphibia, blood gases, Bufo marinus, ApH/At, imidazolealphastat, intracellular pH, Xenopus laevis.

14 R. G. BOUTILIER, M. L. GLASS AND N. HEISLER

findings which suggested that arterial plasma pH changed with temperature in amanner similar to that in neutral water (ApN/At = —0-019 units°C~' from 5 to20°C; ApN/At = -0-017 units "CT1 from 20 to 35°C; Weast, Handbook of Chemis-try and Physics; see Heisler, 19866). However, as more experimental data onindividual animals became available, it was increasingly evident that the magnitudeof changes in pH with temperature (ApH/At) was quite variable, ranging from-0-008 to -0021 in turtles (Robin, 1962; Malan, Wilson & Reeves, 1976), from-0-001 to -0-017 in lizards (Wood, Glass & Johansen, 1977; Wood & Moberly,1970), from -0-011 to -0-021 in amphibians (Mackenzie & Jackson, 1978; Malan etal. 1976) and from -0-008 to -0-017 in fish (Walsh & Moon, 1982; Heisler, 19846;for a complete tabulation of data for all orders of animals see Heisler, 19866).

The alphastat hypothesis of Reeves (1972) predicts that the observed decrease inpH with increasing body temperature (Reeves, 1972; Malan et al. 1976) is based onadjustment towards a constant ionization of histidine—imidazole. As a centralcriterion of the model, Reeves proposed that pH adjustment was accomplished byregulation of ventilation and therefore Pco • However, it was postulated that thebicarbonate concentration would remain constant because of the non-titration ofnon-bicarbonate buffers and the lack of any transmembrane or transepithelial, acid—base-relevant ion transfer. This type of pH regulation was claimed for both theextracellular and intracellular body compartments with pH/temperature coefficients(ApH/At) close to the ApK/At of biological histidine—imidazole moieties[ApKjm/At =—0-018 to — 0-024 units °C~', depending on ligands and steric ar-rangement (Edsall & Wyman, 1958; see Heisler, 19866)]. However, with constantbicarbonate concentration and semi-closed buffer system regulation (open only forCO2, see Heisler, 1986a), constant imidazole dissociation can only be achievedwith histidine—imidazole as the predominant non-bicarbonate buffer. The histidine—imidazole of haemoglobin is the predominant non-bicarbonate buffer of theextracellular space, but intracellular tissue buffering usually deviates considerablyfrom this (Heisler & Neumann, 1980; Heisler, 1984a; see also Heisler, 19866).Nevertheless, the magnitude of ApHe/At is highly variable within and amongstdifferent groups of ectotherms, with most data falling outside the predictions of thealphastat model (see Heisler, 19866).

Considerably less is known about changes of intracellular pH with temperature,even though histidine—imidazole protein residues have been suggested as possibleintracellular sensors for the regulation of the ratio of CO2 production to ventilationand, thus, the regulated variable of the proposed system, Pco (Rahn & Reeves,1980). In fact, with amphibians and reptiles (the animals for which the alphastathypothesis was originally conceived), intracellular pH as a function of temperaturehas been determined in only three species (Malan et al. 1976; Bickler, 1982;P. Neumann, G. M. O. Maloiy & N. Heisler, in Heisler, 19866). In experiments onthe turtle, Pseudemys scripta, ApHj/At values in white muscle ( — 0-0186 units °C~')and liver (—0-023 units°C~') were not significantly different from the range of theApK/At of imidazole compounds and of simultaneously measured blood

Acid—base status and temperature in amphibians 15

(-0-0207units°C~'), whereas ApH,/At of heart (-0-0122 units °C~') was signifi-cantly lower (Malan et al. 1976). In parallel studies on the bullfrog, the ApH/At ofmixed types of striated muscle (—0-015 unitsOC~') was not significantly differentfrom that of arterial blood (-0-0206 units°C~'). More recently, Bickler (1982) foundthat brain intracellular pH of the lizard, Dipsosaurus dorsalis, was maintainedconstant over an 18—35°C temperature range, whereas other tissue compartmentsconformed to the alphastat model at least in the preferred temperature range. As withthe findings of Bickler (1982) for Dipsosaurus brain, the intracellular pH of Varanusexanthematicus white muscle (ApH/At = —0-005 units°C~'), heart (ApH/At =-0-003 unitsX"1) and oesophagus (ApH/At = -0-002units"C"1) changed littlewith changes in temperature (Neumann et al. in Heisler, 19866). From these studies,data on intracellular pH in amphibians are available for only one muscle type in onespecies of Anura (Malan et al. 1976). This scarcity would be of little importance wereit not for the primary role that the amphibian model has played in formulating thealphastat hypothesis (Reeves, 1972). Against this background we have examined thepH/temperature relationships of several tissue types in the aquatic anuran, Xenopuslaevis, and in the semi-terrestrial toad, Bufo marinus.

MATERIALS AND METHODS

Specimens of Bufo marinus (mass 210-423 g, Ar= 27) and Xenopus laevis (mass47—65 g, N = 28) were purchased from a commercial supplier (Charles D. SullivanCo. Inc., Nashville, TN) and transported to Germany by airfreight. Animals wereheld in captivity for at least 1 month prior to experimentation and fed on crickets andchopped liver with vitamin supplements. Ten days before implantation of femoralartery cannulae (see Boutilier, Randall, Shelton & Toews, 1979; Boutilier, 1984)animals were acclimated to either 10, 20 or 30°C in thermostatically controlledaquaria or terraria (±1°C). For cannulation, animals were anaesthetized byimmersion in a 0-7% solution of tricaine methane sulphonate (MS-222, Sigma)titrated to pH 7 with sodium bicarbonate. The entire operation including anaesthesiatook approximately 1 h, whereupon the animals were returned to their respectiveacclimation temperature for a 48-h recovery period. The air and water phases of theexperimental chambers were held to within ±0-5 °C of the acclimation temperatures.

Twelve hours before each experiment, [14C]DMO (5,5-dimethyl-2,4-oxazolidine-dione) and [3H]inulin were injected into the femoral artery for subsequentestimation of intracellular pH by use of the DM0 distribution technique (Waddell &Butler, 1959). Blood samples (about 300//I) were taken from the animals (four fromBufo and two from Xenopus) at their respective acclimation temperatures andanalysed for plasma pH, total CO2 concentration, Pco ar)d Po • I n the case ofXenopus, the samples were taken shortly after a breathing period at the surface (seeBoutilier, 1984). Immediately after the last blood sample (which included anadditional 300 fi\ for plasma isotope analysis) had been taken, the animals wereswiftly killed by anaesthetic overdose and tissue samples removed for subsequentanalysis of tissue water compartments and radioactivity. The gastrocnemius and


sartorius muscles from the non-cannulated limbs were dissected away, as wereindividual muscle bands associated with the pectoral area. Five samples(100-400 mg) from each skeletal muscle group of each animal were taken for analysis.Samples of heart tissue (50-150 mg) were taken from Bufo (five samples) andXenopus (1—2 samples). Immediately after removal, tissues (and correspondingplasma samples) were weighed and then dried to a constant weight (at 110°C) beforebeing prepared, by oxidation, for liquid scintillation counting.

Dried tissue samples and plasma samples were pressed into filter paper pills andcombusted with a sample oxidizer (Packard Instruments, Model 306, modified) foranalysis of [14C]DMO and [ Hjinulin by liquid scintillation counting (PackardInstruments, Model 2660) (see Heisler, 1975, for details). The ratio of theextracellular fluid volume of muscle to the total muscle water, obtained from theconcentrations of [ Hjinulin in muscle and plasma water, was taken to representfractional extracellular volume (Qcm)- Intracellular pH (pH;) of muscle wasdetermined by the transmembrane distribution of [14C]DMO according to theequation:

pH = P K D M O + l o

where pKDMO = 6-464-0-00874t (Albers, Usinger & Spaich, 1971). IntracellularDMO concentrations ([DM0];) were calculated according to the relationship:

r n M O 1 _[DMO] m -[DM0] e xQ e m

L j i - (1-Qem)

where [DM0]m and [DM0]e are the concentrations in the total muscle water andplasma water, respectively.

Measurements of blood pH, Pco and Po were carried out using Radiometer bloodmicro systems (BMS3) calibrated at the respective temperatures with precisionbuffers (Radiometer S1500, S1510) and humidified gas mixtures (Wosthoff gasmixing pumps, Bochum, FRG). The total CO2 concentrations of 50-̂ il samples ofanaerobically obtained true plasma were determined using the electrode and cuvettemethod of Cameron (1971). Bicarbonate concentrations ([HCC>3~]) in true plasmawere estimated from the measured values of total CO2 concentration ([CO2]) andP c o , using the equation:

] - [CO2] - 2

where arCO2 is the solubility of carbon dioxide in plasma (in mmoll" rnmHg"1)(1 mmHg = 1333 Pa) at the respective acclimation temperature (Heisler, 19846,1986a; molarity = 0-223 moll"1; note that the sign of the last line term of the aC02

formula is misprinted in Heisler, 1984, and should read ' + ').Whole blood oxygen dissociation curves were constructed in vitro (at 10, 20 and

30 °C) on blood samples obtained from cannulated animals at each of the respectivetemperatures. Samples of blood were pooled to achieve the required volumes anddistributed equally to tonometers supplied with either air/CO2 or N2/CO2 gas

Acid-base status and temperature in amphibians 17

mixtures (the C02 concentrations being adjusted at each temperature to approximatethe corresponding levels of arterial Pco seen in vivo). Blood samples of known O2saturation were prepared for PQ and pH measurement as described by Hicks,Ishimatsu & Heisler (1987), using the mixing method (Haab, Piiper & Rahn, 1960)detailed by Scheid & Meyer (1978).

Statistical significance of differences between variables was determined byapplication of Student's i-test at a level of 2P<0-05.

RESULTS

The blood pH/temperature relationships (ApHe/At) in both Xenopus laevis andBufo marinus were not significantly different from that of neutral water over theentire temperature range studied (10-30°C) (Fig. 1; Table 1). In both species thedecreases in blood pH with increasing temperature were achieved primarily byincreases in arterial Pco ; the changes in plasma [HCOj"], although statisticallysignificant, contributed less to the change in pH (Fig. 2).

The pH/temperature relationships of skeletal and cardiac muscle tissue compart-ments of Xenopus laevis and Bufo marinus are plotted in Fig. 3. Unlike arterialblood, ApH/At in the intracellular compartments varied considerably with tempera-ture (Table 1) and was often markedly different from that of arterial blood. In Bufomarinus, for instance, ApH/At for skeletal muscle was lower in the temperature

8-0

u 7-i

a.

7-6 \ApN/At= -0-018 \

I20

Temperature (°C)

40

Fig. 1. Relationship between arterial plasma pH (extracellular pH, pHc) and ambienttemperature in Xenopus laevis ( • , x ± S.E., Ar = 10 at 10°C; N = 8 at 20°C; N = 10 at30°C) and Bufo marinus ( • , x±s .E . , A7 = 8 at 10°C; A' = 10 at 20°C; N = 9 at 30°C).Each A' is the mean of two (Xenopus) or four (Bufo) independent measurements.


range 10-20°C than at higher temperatures. However, the reverse was true forXenopus laevis (Table 1). At any given temperature there were no significantdifferences in pH among the three groups of skeletal muscle in Xenopus laevis,

20

00

E

Oa.

10

L L20 40

30

ouX

20

I20

Temperature (°C)40

Fig. 2. Arterial plasma Pcoz and bicarbonate concentration [HCO3 ] as a function ofambient temperature in Xenopus laevis ( • ) and Bufo marinus ( • ) (x ± S.E., N as forFig. 1; S.E. are sometimes covered by the points indicating the average).

Table 1. pH changes with changes in body temperature (ApH/At, units°C ') inbody fluid compartments o/Bufo marinus and Xenopus laevis

Species

Xenopus laevis

Bufo marinus

Body fluidcompartment

Arterial plasmaSartorius muscleGastrocnemius musclePectoral muscleHeart muscle

Arterial plasmaSartorius muscleGastrocnemius musclePectoral muscleHeart muscle

10-20°C

-0018-0017-0-027-0-018

0-000

-0-015-0014-0011-0010-0-024

Temperature range

20-30°C

-0015-0014-0-007-0-011-0-014

-0-014-0-030-0036-0-017-0029

10-30°C

-0-017-0-016-0-017-0-014-0-007

-0015-0022-0-023-0014-0026

Acid-base status and temperature in amphibiansA

7-2

70

19

6-8Xenopus laevis

I I

ApN/At= -0018

X 7

Q.

7-4

7-2

7-0

6-8

0

B

20 40

o Gastrocnemius muscle• Sartorius muscle

Cardiac musclePectoral muscle

Bufo marinus N ApN/At = -0-018

I | I I I0 20

Temperature (°C)

40

Fig. 3. Intracellular pH as a function of ambient temperature for three groups of skeletalmuscle and for cardiac muscle of Xenopus laevis (A) and Bufo marinus (B) (x±S.E.,which is presented in Table 3, has been omitted for clarity; N as for Fig. 1, each A' is themean of five independent measurements, except for heart muscle, 2, of Xenopus).

although pH in the pectoral muscle of Bufo marinus was significantly lower than ineither the sartorius or gastrocnemius muscle at 10 and 20°C (Fig. 3; Tables 2, 3). Inboth species, the pH of cardiac muscle was higher than that of skeletal muscle at anygiven temperature (Fig. 3). There were marked differences in the pH/temperatureresponse of heart tissue between the two species. Cardiac muscle in Bufo marinus(Fig. 3) showed the largest change in pH with temperature of all the compartmentsstudied (Table 1). In Xenopus, however, heart muscle pH was constant in the10-20°C temperature range, although in the 20-30°C range the ApH,/At of heartmuscle tissue was not significantly different from that of arterial plasma (Table 1).

The values for the fractional water content of the tissues (FH Q) and extracellularspace (Qcm), obtained using the inulin distribution technique, are presented in

20

100

3 50

R. G. BOUTILIER, M. L. GLASS AND N. HEISLER

100

50

Xenopus laevis

50 100

PCO,

50 100

Fig. 4. Oxygen dissociation curves for whole blood of Xenopus laevis and Bufo marinusat 10, 20 and 30°C. Each curve was constructed in vitro from blood samples pooled from2-3 animals acclimated for at least 96 h to the respective temperatures. Solid circlesrepresent in vivo arterial blood Pco: values (x ± S.E.) for the respective temperature.

Table 2. Intracellular pH (pHJ, fractional water content (F^2Q), and fractionalextracellular volume (Qem) of several skeletal muscle species and heart muscle of

Xenopus laevis

Parameter

pH,

FH2O

Values are

Temperature(°C)

102030

102030

102030

x ± S.E.

Sartoriusmuscle

7-206 ± 0-0267-033 ±0-0486-893 ± 0-045

0-785 ±0-0050-778 ±0-0080-786 ±0-008

0-145 ±0-0050-155 ±0-0080-162 ±0-005

Gastrocnemiusmuscle

7-243 ± 0-0276-975 ± 0-0476-908 ±0-037

0-785 ± 0-0030-776 ± 0-0080-788 ±0-008

0-159 ±0-0080163 + 00100-145 ±0-003

Pectoralmuscle

7-231 ±0-0257-054 ±0-0386-944 ±0-036

0-790 ±0-0030-756 ±0-0080-788 ±0-010

0-229 ±0-0060-240 ±0-0060-241 ±0-009

Heartmuscle

7-217 ±0-0297-217 ±0-0457-075 ± 0-049

0-852 ±0-0070-813 ±0-0070-813 ±0-007

0-223±0-0110-299 ±0-0260-280 ±0-014

Tables 2 and 3. There was no significant effect of temperature on either measurementin either species.

Arterial blood P o increased with temperature in both species. These Pao valueswere obtained during periods when the animals were ventilating their lungs {Bufomarinus) or shortly after a breathing episode {Xenopus laevis). Whole blood oxygenequilibrium curves, determined in vitro for both species, are shown in Fig. 4 at eachof the acclimation temperatures examined in the present study. Pco levels of the

Acid-base status and temperature in amphibians 21

Table 3. Intracellular pH (pH,), fractional water content (FHlO), and fractionalextracellular volume (Qem) °f several skeletal muscle types and heart muscle of

Bufo marinus

Parameter

pH,

FH2O

Values are

Temperature(°C)

102030

102030

102030

X±S.E.

Sartoriusmuscle

7-418 ± 0 0 3 37-274 ±00316-978 ± 0 0 4 9

0-805 ± 0 0 1 30-786 ±0-0070-791 ±0-005

0-160±00160-202 ± 0 0 1 90-224 ±0-021

Gastrocnemiusmuscle

7-389 ± 0 0 2 67-282 ±0-0276-924 ±0-067

0-799 ± 0 0 0 60-779 ±0-0040-786 ±0-003

0-111 ± 00090145 ±0-0160-149 ±0-004

Pectoralmuscle

7-281 ±00467-178 ±0-0407-004 ±0-034

0-815 ±00080-797 ±00030-805 ± 0005

0-201 ±00180-223 ± 00200-1% ±0029

Heartmuscle

7-5% ±00387-353 ±0-0497-068 ±0078

0-815 ±0-0060-808 ±0-0060-817 + 0-004

0-225 ±0-0060-257 ±0-0230-287 ±0-040

equilibrating gas mixtures were set close to the values obtained in vivo at therespective temperatures (Fig. 2), resulting in a pH-induced shift to the right withincreasing temperature. When the measured values of Pao at each acclimationtemperature in vivo are plotted on their corresponding O2 equilibrium curve (Fig. 4)it is evident that arterial blood O2 saturation remains relatively constant over theentire temperature range.

DISCUSSION

The changes in arterial blood Po with temperature in Xenopus and Bufo occur at arelatively constant arterial blood O2 saturation (Fig. 4). This can be explained on thebasis of existing models for O2 transport in animals with cardiovascular shunts(Wood, 1982, 1984; Wood & Hicks, 1985). Arterial desaturation during air breathingresults from systemic venous admixture with oxygenated pulmonary venous blood(right to left intraventricular shunt; Shelton, 1976; Shelton & Boutilier, 1982).Under these conditions, the arterial Po of a mixture of pulmonary and mixed venousblood becomes a function of the resulting O2 saturation (Wood, 1982). Therightward shift of the oxygen dissociation curves with rising temperature (Fig. 4)results, therefore, in an increase in arterial P o with temperature. Within thetemperature range examined in the present study, the arterial O2 saturation ofXenopus (during or shortly after a breathing episode) was maintained virtuallyconstant, whereas in Bufo a small but progressive desaturation occurred astemperature increased (Fig. 4). Such effects have been previously demonstrated in anumber of reptiles (Wood, 1982; Glass, Boutilier & Heisler, 1983; Wood & Hicks,1985) and in two amphibians (Wood, 1982; Kruh0ffer, Glass, Abe & Johansen,1987). Although the underlying mechanisms of such phenomena are yet to bedetermined, the collected data imply that the common denominator of oxygenhomeostasis in ectotherms may be O2 saturation and/or content, rather than partial


pressure (Glass et al. 1983; Wood & Hicks, 1985). Intraventricular shunting andvenous mixture has less of an effect on CO2 partial pressures owing to differentcharacteristics of the CO2 dissociation curves.

The arterial blood pH values of Bufo marinus and Xenopus laevis vary inverselywith temperature (ApH/At = — 0-015 and —0-017, respectively; Fig. 1; Table 1,range 10—30°C). These values are similar to those reported for the arterial blood ofother amphibian species (Howelle* al. 1970; Reeves, 1972; Burggren & Wood, 1981;Moalli, Meyers, Ultsch & Jackson, 1981; Hicks & Stiffler, 1984; Kruh0ffer et al.1987). The changes in arterial pH with temperature in the present study are broughtabout primarily through adjustments in Paco 1 though small but significant changesin plasma bicarbonate concentrations with temperature were also observed (Fig. 2).In all instances, it appears that the arterial blood pH of amphibians varies inverselywith temperature by a specific constant from the pH of neutral water, or that theanimals maintain a constant relative alkalinity (Rahn, 1967). The compiled data onarterial blood of amphibians also fit the alphastat hypothesis of Reeves (1972). Thebiological importance of this hypothesis rests, however, on the assumption that thepH values of intracellular compartments also change with temperature, more or lessin parallel with that of arterial blood (constant relative acidity).

The pH,/temperature relationships for skeletal and cardiac muscle in the presentstudy do not follow the patterns predicted by the alphastat model (Fig. 3; Table 1).Previous measurements of intracellular pH in amphibians are restricted to one studyon Rana catesbeiana (Malan et al. 1976) in which the pH, of striated muscle(apparently combinations of gracilis, sartorius and gastrocnemius) was determinedas a function of temperature by the DM0 method. Malan et al. (1976) foundApH,/At in striated muscle (—0-0152units°C~l) to be not significantly differentfrom that of arterial blood (—0-0206 units °C~'). In our study, average ApHj/Atvalues for the range 10—30°C varied in different groups of striated muscle from— 0-014 to —0-023 for Bufo marinus and from —0-014 to —0-017 for Xenopus laevis(Table 1), the differences being even larger for smaller temperature intervals.ApH/At tended to be lower at lower temperatures in Bufo, whereas the reverse wastrue for Xenopus laevis. These data suggest that intracellular pH may vary withtemperature in a non-linear fashion, a conclusion also reached by Walsh & Moon(1982) on the basis of data obtained by acid-base/temperature studies in the eel.Similar non-linearities can also be found for the extracellular fluid of reptiles (forreferences see Heisler, 19866). They are, however, not universal. For example, theApH/At of arterial plasma of both species is more or less constant, as is ApH,/At ofBufo heart muscle and Xenopus sartorius muscle. In all of the other intracellular bodycompartments, however, there is a remarkable difference in ApH/At between thelower and higher temperatures (Fig. 3; Table 1). This is particularly pronounced inheart muscle of Xenopus, where pHj is held constant over the 10—20°C range, butfalls by 0-14pH units between 20 and 30°C (Fig. 3; Tables 1, 2). Similarobservations were made in eel heart muscle (Walsh & Moon, 1982). These dataindicate that mechanisms other than simple physicochemical dissociation processesare responsible for the fine adjustment of pH with changes of temperature.


Accordingly, there is no good a priori reason to assume that the pH/temperaturerelationship of any body compartment should follow a linear pattern.

Over the past few years, discussions about pH/temperature relationships inectotherms and the validity of the alphastat hypothesis have become reduced toarguments about the relative magnitude of linearly interpreted ApH/At slopes. Thepresent data on intracellular pH changes with temperature support data from theliterature on non-amphibian ectotherms, showing a wide range of physiologicalApH,/At values in various body compartments (see Heisler, 19866). These data alsoshow that temperature-related intracellular acid-base regulation is relatively inde-pendent of extracellular acid-base regulation in amphibians, and clearly indicate thatindividual compartments may maintain unique pH levels and unique ApH/Atrelationships (see Fig. 3). The alphastat model, with adjustment of only organismicPco > cannot account for regulatory patterns such as these. Although less restrictedthan in fishes (see Heisler, 19846), adjustment of Pco m intermittently breathinganimals is limited by the discontinuity of gas exchange (Boutilier & Shelton, 1986;Toews & Boutilier, 1986), and the resulting isothermal variability in arterial bloodPco is likely also to be transmitted to intracellular body compartments. IntracellularpH regulation is, accordingly, passively affected by changes in Pco and thetemperature-dependent buffering characteristics, which are quite variable amongspecies and among different tissues in individual species (see Heisler, 1986a,b). Theactive regulatory process, however, has to be sought in transmembrane andtransepithelial ion transfer mechanisms. It should be emphasized that evenconcordance of ApH/At with ApK/At values of imidazole moieties (a relatively rarecoincidence) (for references see Heisler, 19866) provides little support for thealphastat model, if production of acid-base-relevant ions (by metabolic andbuffering processes) and transfer of such ions across the borders of the respectivecompartments are neglected. Future studies must not, therefore, be limited todescriptions of pH/temperature coefficients, but should focus on detailed evaluationof more indicative features of acid-base regulation.

The authors gratefully acknowledge the skilful technical assistance of Mrs S.Glage and Mr G. Forcht. Supported by Alexander von Humboldt Stiftung andDeutsche Forschungsgemeinschaft.

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